Lentiviral based gene transfer systems represent a promising gene delivery technology due to their ability to efficiently transduce a variety of non-dividing target cells in vitro and in vivo. However, most data are from studies using Human Immunodeficiency Virus (HIV), the causative agent of AIDS. Advanced Vision Therapies, Inc. (AVT) has developed a novel lentiviral vector system based on bovine immunodeficiency virus (BIV), an animal lentivirus not associated with any known human disease. Our preliminary data showed that VSV-G pseudotyped BIV vector efficiently transduced a variety of non-dividing cells in vitro and retinal and neuronal cells in vivo, leading to sustained transgene expression. Furthermore, BIV vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in vivo, suggesting that the vector is suitable for clinical applications. AVT is currently applying this vector system to the treatment of human sight-threatening eye diseases. One of the major limitations to the clinical application and commercialization of lentiviral vectors is the lack of a stable vector packaging cell line for efficient, predictable vector production, scale-up and manufacturing. Currently, calcium phosphate mediated transient transfection is being used for lentiviral vector production, which is not ideal for clinical use. The inability to generate a stable packaging cell line is due to the fact that two components required for the cell line development, Gag/Pol and VSV-G Env, are cytotoxic and cannot be constitutively expressed in a cell line. The goal of this Phase I study is to develop a stable packaging cell line that constitutively produces high titer BIV-based lentiviral vectors and that is amenable to scale-up and manufacturing. There are three specific aims: 1. To identify and eliminate nuclear retention sequences in the vector to generate a gene transfer system that is independent of Rev/RRE; 2. To verify that BIV-based vectors pseudotyped with the Baculovirus GP64 envelope protein efficiently transduce retinal cells in vivo; and 3. To generate a stable lentiviral vector packaging cell line. Completion of specific aim 1 will improve the vector safety profile by eliminating the viral RRE sequence from the vector and will facilitate packaging cell line generation by eliminating the need for Rev. Specific aim 2 is also designed to facilitate packaging line development. AVT has previously solved the problem of Gag/Pol cytotoxicity and has engineered cell lines that constitutively express these proteins. Completion of specific aim 2 will solve the problem of envelope cytotoxicity by enabling the use of the GP64 envelope. Finally, the creation of a packaging cell line in specific aim 3 will overcome the most critical, rate-limiting step in applying lentiviral vectors to the treatment of human disease.